Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase

Abstract

Mrc1 is a conserved checkpoint mediator protein that transduces the replication stress signal to the downstream effector kinase. The loss of mrc1 checkpoint activity results in the aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early origin firing in a checkpoint-independent manner, but its mechanism was unknown. Here we identify HBS (Hsk1 bypass segment) on Mrc1. An ΔHBS mutant does not activate late/dormant origin firing in the presence of hydroxyurea but causes the precocious and enhanced activation of weak early-firing origins during normal S-phase progression and bypasses the requirement for Hsk1 for growth. This may be caused by the disruption of intramolecular binding between HBS and NTHBS (N-terminal target of HBS). Hsk1 binds to Mrc1 through HBS and phosphorylates a segment adjacent to NTHBS, disrupting the intramolecular interaction. We propose that Mrc1 exerts a "brake" on initiation (through intramolecular interactions) and that this brake can be released (upon the loss of intramolecular interactions) by either the Hsk1-mediated phosphorylation of Mrc1 or the deletion of HBS (or a phosphomimic mutation of putative Hsk1 target serine/threonine), which can bypass the function of Hsk1 for growth. The brake mechanism may explain the checkpoint-independent regulation of early origin firing in fission yeast.KEYWORDS DNA replication timing, Mrc1, Hsk1, intramolecular interaction, replication checkpoint I n eukaryotic cells, DNA replication is initiated from a number of sites (replication origins) (1, 2). In fission yeast, more than 1,000 prereplicative complexes (pre-RCs) are generated on chromosomes during G 1 phase, and only a subset of them are used for initiation (3-9). The choice of origins to be fired and the determination of the timing of their activation are influenced by many factors, including chromatin structures, local histone modifications, transcription factors, and even physiological conditions such as temperature (7,(10)(11)(12)(13)(14)(15)(16)(17).The DNA replication checkpoint (intra-S checkpoint) operates to delay the activation of late-firing/dormant origins in response to an inhibition of DNA synthesis. The conserved Mec1-Mrc1-Rad53/Rad3-Mrc1-Cds1/ATR-Claspin-Chk1 pathway plays a major role in mediating the replication checkpoint signal to the downstream pathway. Mrc1/Claspin plays a major role as a mediator for the activation of this pathway. We previously reported that Hsk1 is required for the activation of the replication checkpoint, probably through the phosphorylation of Mrc1 (18). On the other hand, Mrc1 was also shown to be required for the scaling of DNA replication timing in budding yeast. In most mutants that extend the S phase, the activation timings of most origins are delayed in proportion to the duration of S phase. In contrast, early-firing origins fire early, even in the extended S phase, in ΔMRC1 cells (19). Importantly, this requirement of Mrc1 for the scaling of origin activation could...

The Protective Role of Dormant Origins in Response to Replicative Stress

“…Also, Claspin plays another role in the initiation of DNA replication in human cells during the normal S phase by recruiting Cdc7 to facilitate phosphorylation of MCM proteins [ 136 ]. It was recently discovered in yeast that Mrc1 has two crucial functions in regulating the firing of origins: a checkpoint independent-role to activate early-firing origins during normal replication, and a checkpoint dependent-function to inhibit late/dormant origins in the presence of HU [ 137 ].…”

Implications of CLSPN Variants in Cellular Function and Susceptibility to Cancer

“…It has a ring-shaped DNA binding structure that shows high affinity for replication associated structures [ 21 , 22 ] and interacts with numerous replication proteins [ 21 , 22 , 23 , 24 , 25 ], being an integral component of the replisome [ 5 , 21 , 22 , 24 , 26 ]. Claspin is required for ensuring normal rates of replication [ 27 , 28 ] and functions together with Cdc7 to regulate origin firing [ 29 , 30 ]. Replication fidelity is of paramount importance for the maintenance of genome integrity.…”

The Protecting Role of Dormant Origins in Response to Replicative Stress

“…A new role for Claspin has been described more lately in the initiation of DNA replication during normal S phase through the recruitment of CDC7 kinase that facilitates phosphorylation of MCM proteins [131]. Besides its checkpoint function, it has been recently discovered that the loss of MRC1 checkpoint activity leads to aberrant activation of late or dormant origins in the presence of replication inhibitors HU [132]. MRC1, with these two crucial functions, can be placed at the heart of origin firing regulation: one regulating late/dormant origins through its well-established checkpoint function and the other regulating early-firing origins through checkpoint-independent mechanism [132].…”